The present invention relates to compositions derived from immunoglobulin molecules specific for the hepatitis C virus (HCV). More particularly, the invention is related to recombinant human monoclonal antibodies which are capable of specifically binding with HCV E2 antigen.
Hepatitis C virus (HCV) infection occurs throughout the world and is the major cause of transfusion-associated hepatitis. There are an estimated 150,000 new cases of HCV infection each year in the United States. The seroprevalence of anti-HCV antibodies in blood donors from around the world has been shown to vary between 0.02 and 1.23%, with rates in some countries as high as about 19%. In addition to being the predominate cause of transfusion-induced hepatitis, HCV is also a common cause of hepatitis in individuals exposed to blood or blood products. Thus, recipients of blood or blood products, intravenous drug users, renal dialysis patients and needle-stick victims represent high-risk groups for HCV infection. Alter et al. (1993) Infect Agents Dis 2:155-166. Further, heterosexual transmission of HCV across the urogenital tract, and mother-to-baby transmission, has been well documented. Ohto et al. (1994) N Engl J Med 330:744-750. Other risk factors associated with HCV infection include familial or household contact with an HCV-infected individual and health-care employment with occupational exposure to blood and hemodialysis. Alter et al. (1990) JAMA 264:2231-2235. Chronic hepatitis develops in approximately 62% of infections. Alter et al. (1992) N Engl J Med 327:1899-1905.
Most of the serious liver disease associated with HCV results from the high propensity of the agent to cause chronic, persistent infection. Cirrhosis occurs in approximately 20% of chronic cases, of which 20 to 25% will result in liver failure. Another serious sequela associated with HCV infection is primary hepatocellular carcinoma.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. Currently, there are 6 distinct, but related genotypes of HCV which have been identified based on phylogenetic analyses (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polypeptide having more than 3000 amino acid residues (Choo et al. (1989) Science 244:359-362; Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455; Han et al. (1991) Proc. Natl. Acad. Sci. USA 88:1711-1715). The polypeptide is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, there are three putative HCV structural proteins, consisting of the N-terminal nucleocapsid protein (termed xe2x80x9ccorexe2x80x9d) and two envelope glycoproteins, xe2x80x9cE1xe2x80x9d (also known as E) and xe2x80x9cE2xe2x80x9d (also known as E2/NS1). (See, Houghton et al. (1991) Hepatology 14:381-388, for a discussion of HCV proteins, including E1 and E2.) E1 is detected as a 32-35 kDa species and is converted into a single endo H-sensitive band of approximately 18 Kda. By contrast, E2 displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species (Grakoui et al. (1993) J. Virol. 67:1385-1395; Tomei et al. (1993) J. Virol. 67:4017-4026). The HCV envelope glycoproteins E1 and E2 form a stable complex that is coimmunoprecipitable (Grakoui et al. (1993) J. Virol. 67:1385-1395; Lanford et al. (1993) Virology 197:225-235; Ralston et al. (1993) J. Virol. 67:6753-6761).
The only currently available treatment for chronic hepatitis C infection consists of xcex1-interferon (xcex1-IFN) therapy. However, long-term response to interferon therapy only occurs in 10% to 30% of treated individuals, and there is evidence that the different HCV strains vary greatly in their responsiveness to interferon therapy, with the type 1 viruses being the most refractive. Furthermore, flu-like side effects are commonly encountered with interferon therapy (occurring in approximately 60% to 80% of treated individuals), as well as other less common side effects such as nausea, depression, fatigue and thrombocytopenia. Interferon therapy is also not indicated for immunocompromised individuals. Accordingly, there exists a need for more effective therapeutic approaches in the treatment of chronic HCV infection. In this regard, some effect has been seen using ribivirin, or combination therapies with ursodiol and xcex1-IFN.
In particular, the HCV E1 and E2 proteins are of considerable interest because recombinant vaccines based on those molecules have been shown to be protective against experimental challenge with HCV in primate studies. (Choo et al. (1994) Proc. Natl. Acad. Sci. USA 91:1294-1298). Hyperimmune globulin compositions of anti-HCV antibody molecules obtained from donor samples have been described for the treatment of HCV in infected individuals, and in the prevention of HCV infection in high-risk groups. European Patent Application Publication No. 447,984, published Sep. 25, 1991. Since these compositions are made from donor blood products, an inherent risk is associated with their use due to the possible presence of infectious against such as the Human Immunodeficiency Virus (HIV) and HCV. Accordingly, hyperimmune globulin preparations must be carefully screened, and all infectious agents inactivated prior to administration to human subjects.
It is known that the immune response to HCV in normal individuals includes both humoral and cell mediated components. Koziel et al. (1993) J Virol 67:7522-7532, Alter et al. (1989) N Engl J Med 321:1494-1500. Further, several reports have indicated that antibodies elicited to HCV may neutralize the infectivity of the virus. Shimizu et al. (1994) J Virol 68:1494-1500, Farci et al. (1994) Proc Natl Acad Sci USA 91:7792-7796. Such results provide hope that an effective antibody-based therapy can be developed. In this regard, the administration of a highly-reactive, neutralizing anti-HCV antibody preparation to an individual who is at risk of infection, or who has been recently exposed to the agent will provide immediate passive immunity to the individual. Such passive immunizations would likewise be expected to be successful in both normal and immunocompromised subjects. Preferably, the neutralizing antibodies would be broadly cross-reactive against different HCV strains, and would be monoclonal in order to control the effects of the use of the antibodies in vivo.
For a number of practical and economic reasons, murine monoclonal antibodies have been generally used in research and medicine. Murine antibodies can be raised against a wide variety of molecules, such as HCV antigens, and fused with a myeloma cell to yield hybridomas which can be grown in culture to produce monoclonal antibodies toward HCV antigens. Kohler et al. (1975) Nature 256:495-497. Although such monoclonal antibodies may have antigen binding specificities of significant therapeutic value, the use of such murine antibodies in the treatment of human disease has been limited since those molecules are immunogenic to the human immune system. Thus, murine monoclonals have been most commonly used in immunodiagnostics. In this regard, murine monoclonal antibodies to putative HCV E2 envelope polypeptides have been described for use in the detection of HCV in biological samples. U.S. Pat. No. 5,308,750 to Mehta et al.
Accordingly, there remains a need in the art to provide human monoclonal antibodies toward HCV E2 antigen, wherein the monoclonals are broadly cross-reactive with heterologous HCV isolates.
The present invention is based on the discovery of human monoclonal antibody molecules which exhibit immunological binding affinity for HCV E2 polypeptide antigen, and which are cross-reactive against different HCV strains. The monoclonal antibody molecules were obtained from a combinatorial library that was constructed from a nonimmunized HCV-infected source. The present molecules generally comprise a human antibody Fab molecule that exhibits immunological binding affinity for HCV E2 antigen.
Accordingly, in one embodiment, the invention is directed to a recombinant human monoclonal antibody that exhibits immunological binding affinity for HCV E2 antigen, wherein the monoclonal antibody includes amino acid sequences that are homologous to the binding portion of a human antibody Fab molecule obtained from a combinatorial antibody library. The recombinant monoclonal antibody molecule can be in the form of a substantially whole immunoglobulin molecule, or can be in the form of a soluble Fab molecule, an Fv fragment, or an sFv molecule, wherein each molecule at least contains amino acid sequences that are homologous to the binding portion of a human antibody Fab molecule.
In another embodiment, the invention is directed to an isolated nucleic acid molecule which contains a polynucleotide coding sequence for a polypeptide that is homologous to the binding portion of a heavy or light chain variable region (VH or VL) of a human Fab molecule which exhibits immunological binding affinity for HCV E2 antigen. In a related embodiment, the invention is directed to an isolated nucleic acid molecule which contains polynucleotide coding sequences for a first polypeptide and polynucleotide coding sequences for a second polypeptide, wherein the first polypeptide is homologous to the binding portion of a heavy chain variable region (VH) of a human Fab molecule which exhibits immunological binding affinity for HCV E2 antigen, and the second polypeptide is homologous to the binding portion of a light chain variable region (VL) of a human Fab molecule which exhibits immunological binding affinity for the HCV E2 antigen.
In other embodiments, the invention pertains to expression vectors comprising the nucleic acid molecules above operably linked to control sequences that direct the transcription of the polynucleotide coding sequences when the vector is present in a host cell or under suitable conditions for the transcription and translation of the polynucleotide coding sequences. Yet further embodiments of the invention pertain to host cells transformed with the vectors of the invention, and methods for producing recombinant polypeptides using the transformed host cells.
In another embodiment, the invention is directed to vaccine compositions comprising the recombinant monoclonal antibody molecules of the invention. Still further embodiments relate to methods of using the vaccine compositions, wherein the vaccines are used to provide an antibody titer to HCV in a mammalian subject, and/or used to provide passive immunity against HCV infection in a vaccinated subject. In related embodiments, the vaccine compositions are used in combination with known anti-HCV therapeutics.
In still further embodiments, the recombinant monoclonal antibody molecules of the invention are used to provide binding complexes which are labeled with a detectable moiety. The labeled binding complexes are used in related embodiments of the invention, such as in specific binding assay methods, for detecting the presence of HCV particles in samples suspected of containing HCV and in specific binding assays for monitoring the progress of anti-HCV treatment of HCV-infected subjects.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.